The Preparation of Electroporation Competent Bacteria
1. Take the attenuated Salmonella typhimurium VNP20009 preserving in glycerol to inoculate
in 1 mL
LB
liquid medium and culture it overnight at 37°C.
2. Inoculate the obtained bacterial solution in antibiotic-free LB liquid medium at 1%, in sure that the final volume is 100 mL. Culture the bacteria to OD600=0.4~0.5 (37°C, 180 rpm), and collect for later use.
3. Pre-cool the bacterial solution for 10 min and sterilized 10% sterile glycerol in advance by ice bath. Centrifuge the bacterial solution at 4°C and 5000 rpm for 5 min, and collect the bacteria precipitation.
4. Wash the precipitate 2 or 3 times with 10% glycerol solution at 1/10 volume of bacteria solution, centrifuge at 4°C and 5000 rpm for 5 min to collect the precipitation.
5. Resuspend the precipitation with 10% glycerol at 1/100 volume of bacteria solution, and divide into 50 μL per tube.
2. Inoculate the obtained bacterial solution in antibiotic-free LB liquid medium at 1%, in sure that the final volume is 100 mL. Culture the bacteria to OD600=0.4~0.5 (37°C, 180 rpm), and collect for later use.
3. Pre-cool the bacterial solution for 10 min and sterilized 10% sterile glycerol in advance by ice bath. Centrifuge the bacterial solution at 4°C and 5000 rpm for 5 min, and collect the bacteria precipitation.
4. Wash the precipitate 2 or 3 times with 10% glycerol solution at 1/10 volume of bacteria solution, centrifuge at 4°C and 5000 rpm for 5 min to collect the precipitation.
5. Resuspend the precipitation with 10% glycerol at 1/100 volume of bacteria solution, and divide into 50 μL per tube.
Electroporation
1. Take 2-3 μL of plasmid (the concentration of plasmid higher than 150 ng/μL), gently mix with 50 μL of
ice-cold competent bacteria suspension, add it to a pre-cooled electroporation cup with a distance of 0.1
cm, put it on ice and stand for 5 min, the setting conditions of the electroporator are 1800 V, 200 Ω, 25
μF; Or add to a pre-cooled electroporation cup with a spacing of 0.2 cm, put it on ice and stand for 5
min, set the conditions to 2500~3000 V, 200 Ω, 25 μF.
2. After the electroporation, quickly suck the liquid in the electroporation cup into the centrifuge tube, add 800 μL of LB liquid medium, culture the bacteria for 1.5 h (37°C, 180 rpm), centrifuge at 25°C and 5000 rpm for 5 min, discard the excess supernatant, remain 100 μL supernatant to resuspend the bacteria precipitation, coat on LB solid medium with Amp antibiotic, incubate overnight at 37°C, and screen positive monoclonals.
3. After the experiment, the electroporation cup needs to be cleaned: first rinse with ddH2O, then rinse with 75% sprinkles, and finally soak in 75% alcohol. Usually, the electroporation cup is soaked in 75% alcohol. Before using, the electroporation cup needs ultraviolet irradiation for 15~30 minutes in clean bench.
2. After the electroporation, quickly suck the liquid in the electroporation cup into the centrifuge tube, add 800 μL of LB liquid medium, culture the bacteria for 1.5 h (37°C, 180 rpm), centrifuge at 25°C and 5000 rpm for 5 min, discard the excess supernatant, remain 100 μL supernatant to resuspend the bacteria precipitation, coat on LB solid medium with Amp antibiotic, incubate overnight at 37°C, and screen positive monoclonals.
3. After the experiment, the electroporation cup needs to be cleaned: first rinse with ddH2O, then rinse with 75% sprinkles, and finally soak in 75% alcohol. Usually, the electroporation cup is soaked in 75% alcohol. Before using, the electroporation cup needs ultraviolet irradiation for 15~30 minutes in clean bench.
Infection of Tumor Cells with the Engineered Bacteria VNP20009
1. Observe the degree of convergence of tumor cells under a microscope, digest the cells in the biosafety
cabinet and count the cells under the inverted microscope.
2. Inoculate the tumor cells in 24-well plates (5×104 cells/well), culture them in 500 μL DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, and observe the cell apposition on the following day.
3. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion, culture at 37°C, 220 rpm for 12 h.
4. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5.
5. Centrifuge the bacterial solution at 4000 rpm for 5 min, and carefully remove the supernatant.
6. Resuspend the precipitate with 500 μL PBS, centrifuge at 4000 rpm for 5 min, and carefully remove the supernatant.
7. Resuspend the precipitate with 500 μL DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, centrifuge at 4000 rpm for 5 min, and remove the supernatant carefully.
8. Resuspend the bacteria with DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, and then divided into four tubes for dilution, inoculated in 24-well plates according to the ratio of cells/bacteria = 1:30/1:150/1:300 (to find the appropriate multiplicity of infection, MOI), and co-cultured for about 5 h. Observe the state of the cells under the microscope.
9. Cells are washed twice with PBS, replaced with complete medium, and continued to incubate for 36 h.
10. If observing with fluorescence microscope, there is no need to wait for protein expression.
2. Inoculate the tumor cells in 24-well plates (5×104 cells/well), culture them in 500 μL DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, and observe the cell apposition on the following day.
3. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion, culture at 37°C, 220 rpm for 12 h.
4. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5.
5. Centrifuge the bacterial solution at 4000 rpm for 5 min, and carefully remove the supernatant.
6. Resuspend the precipitate with 500 μL PBS, centrifuge at 4000 rpm for 5 min, and carefully remove the supernatant.
7. Resuspend the precipitate with 500 μL DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, centrifuge at 4000 rpm for 5 min, and remove the supernatant carefully.
8. Resuspend the bacteria with DMEM medium containing 10% fetal bovine serum without penicillin and streptomycin, and then divided into four tubes for dilution, inoculated in 24-well plates according to the ratio of cells/bacteria = 1:30/1:150/1:300 (to find the appropriate multiplicity of infection, MOI), and co-cultured for about 5 h. Observe the state of the cells under the microscope.
9. Cells are washed twice with PBS, replaced with complete medium, and continued to incubate for 36 h.
10. If observing with fluorescence microscope, there is no need to wait for protein expression.
scFv Experiments: The Experiments of scFv Surface Presentation Technique
Prepare Cells for Infection:
1. Place CEA-positive NUGC-3 cells and CEA-negative BGC-823 cells in a 37°C, 5% CO2 incubator for
culture
(use company-supplied medium for culturing NUGC-3 cells, and DMEM complete medium for culturing BGC-823
cells. RPMI-1640 medium for culturing NUGC-3 cells does not need to prepare antibiotic cell culture medium
in advance).
2. Perform cell counting, inoculate these two kinds of cells in different wells of 24-well plate at 5×104 cells per well, and set up a control group. Culture the cells overnight.
2. Perform cell counting, inoculate these two kinds of cells in different wells of 24-well plate at 5×104 cells per well, and set up a control group. Culture the cells overnight.
Prepare Bacteria for Infection:
1. Take 10 μL of engineered VNP20009 (express Lpp-OmpA-scFv) from the glycerol preservation, and add to 1 mL
LB liquid medium for expansion (37°C, 200 rpm, 12 h).
2. Transfer 50 μL of bacterial solution into 5 mL of LB liquid medium containing Amoxicillin antibiotic (1:100) in a 12 mL bacteria-culturing tube, and culture at 37℃ until OD600 =0.5 (OD600=0.5 equals to approximately 1× 108 CFU/mL).
3. Wash the bacteria prepared to infect BGC-823 cells once with 1×PBS, then wash twice with antibiotic-free DMEM medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free DMEM medium containing 10% FBS; Wash the bacteria prepared to infect NUGC-3 cells once with 1×PBS, then wash with antibiotic-free RPMI-1640 medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free RPMI-1640 medium containing 10% FBS.
2. Transfer 50 μL of bacterial solution into 5 mL of LB liquid medium containing Amoxicillin antibiotic (1:100) in a 12 mL bacteria-culturing tube, and culture at 37℃ until OD600 =0.5 (OD600=0.5 equals to approximately 1× 108 CFU/mL).
3. Wash the bacteria prepared to infect BGC-823 cells once with 1×PBS, then wash twice with antibiotic-free DMEM medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free DMEM medium containing 10% FBS; Wash the bacteria prepared to infect NUGC-3 cells once with 1×PBS, then wash with antibiotic-free RPMI-1640 medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free RPMI-1640 medium containing 10% FBS.
Infection:
1. Replace the complete medium containing antibiotics with antibiotic-free cell culture medium containing
10% FBS (DMEM medium for BGC-823 cells and RPMI-1640 medium for NUGC-3 cells) and culture the cells at 37°C
and 5% CO2 for 30 min.
2. Inoculate the cells with bacteria (Multiplicity of infection, MOI=1:100) by adding the treated bacterial solution to the cell culture medium.
3. Incubate the cells at 37°C and 5% CO2 for 2 hours.
4. Aspirate the culture medium and wash twice with 1× PBS.
5. Add fresh medium containing 100 μg/mL gentamicin and incubate at 37°C and 5% CO2 for 2 hours.
6. Replace the medium containing 100 μg/mL of gentamicin with fresh medium containing 20 μg/mL of gentamicin for 1 hour.
7. Add 16 μg/mL of tetratoxum and continue to incubate the cells.
8. Observe the results of bacterial targeting under the fluorescence microscope after incubating the cells for 5 min, 1 h and 2 h.
2. Inoculate the cells with bacteria (Multiplicity of infection, MOI=1:100) by adding the treated bacterial solution to the cell culture medium.
3. Incubate the cells at 37°C and 5% CO2 for 2 hours.
4. Aspirate the culture medium and wash twice with 1× PBS.
5. Add fresh medium containing 100 μg/mL gentamicin and incubate at 37°C and 5% CO2 for 2 hours.
6. Replace the medium containing 100 μg/mL of gentamicin with fresh medium containing 20 μg/mL of gentamicin for 1 hour.
7. Add 16 μg/mL of tetratoxum and continue to incubate the cells.
8. Observe the results of bacterial targeting under the fluorescence microscope after incubating the cells for 5 min, 1 h and 2 h.
GOx Experiments: The Experiment of Generation of H2O2 to Promote Fenton Reaction
Prokaryotic Expression Verification Experiments
1. Separately take 10 μL of VNP20009 bacteria and 10 μL of SopE-FLAG-GOx engineered VNP20009 bacteria from
the glycerol preservation, and add to 5 mL LB liquid medium for expansion (30°C, 200 rpm, 12 h).
2. Take out 100 μL of bacterial solution from the overnight culture bacteria solution, centrifuge the rest of the bacterial solution at 4000 rpm for 5 min. Discard the supernatant, precipitate and break, and carry out Western Blot experiment.
2. Take out 100 μL of bacterial solution from the overnight culture bacteria solution, centrifuge the rest of the bacterial solution at 4000 rpm for 5 min. Discard the supernatant, precipitate and break, and carry out Western Blot experiment.
Type III secretion system
(T3SS) Functional Verification Experiments
1. Culture BGC-823 cells in 24-well plates, 5×105 cells per well.
2. Separately take 10 μL of VNP20009 bacteria and 10 μL of SopE-FLAG-GOx engineered VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion (30°C, 200 rpm, 12 h).
3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5.
4. After the cells are passsaged for 12 h, the VNP20009 and SopE-FLAG-GOx engineered VNP20009 bacteria are added to the cell culture system at a MOI ratio of 1:150, and coculture 5 h. And then wash the cells 3 times with sterile PBS, complete DMEM medium containing 5% fetal bovine serum and 50 μg/mL gentamicin was added to continue the culture for 24 h.
5. Aspirate the supernatant of the medium, break the cells, and perform Western Blot experiments.
2. Separately take 10 μL of VNP20009 bacteria and 10 μL of SopE-FLAG-GOx engineered VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion (30°C, 200 rpm, 12 h).
3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5.
4. After the cells are passsaged for 12 h, the VNP20009 and SopE-FLAG-GOx engineered VNP20009 bacteria are added to the cell culture system at a MOI ratio of 1:150, and coculture 5 h. And then wash the cells 3 times with sterile PBS, complete DMEM medium containing 5% fetal bovine serum and 50 μg/mL gentamicin was added to continue the culture for 24 h.
5. Aspirate the supernatant of the medium, break the cells, and perform Western Blot experiments.
Function Verification
Experiments
1. Measure Hydrogen Peroxide Level:
Bacterial infection is carried out according to the above bacterial infection experimental protocol (T3SS Work verification experiment step 1~4), and then the medium and cells are taken separately, and hydrogen peroxide detection is carried out separately using the hydrogen peroxide detection kit.
2. Measurement of Cell Viability:
According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8 kit is used to detect cell viability. The absorbance of A450 nm is determined under a microplate reader by incubating with cells for 2 h under a co-incubation condition under a light shield.
3. Measure Lipid Peroxidation Level:
According to the above bacterial infection experimental protocol (T3SS Work verification experiment steps 1~4), bacterial infection is performed, the medium is removed, cells are taken, and lipid peroxidation detection is performed using the lipid peroxidation detection kit.
4. Measurement of Cell Viability treated with Fer-1:
According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8 kit is used to detect cell viability. The cells are treated with Fer-1 in this experiment. The absorbance of A450 nm is determined under a microplate reader by incubating with cells for 2 h under a co-incubation condition under a light shield.
Bacterial infection is carried out according to the above bacterial infection experimental protocol (T3SS Work verification experiment step 1~4), and then the medium and cells are taken separately, and hydrogen peroxide detection is carried out separately using the hydrogen peroxide detection kit.
2. Measurement of Cell Viability:
According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8 kit is used to detect cell viability. The absorbance of A450 nm is determined under a microplate reader by incubating with cells for 2 h under a co-incubation condition under a light shield.
3. Measure Lipid Peroxidation Level:
According to the above bacterial infection experimental protocol (T3SS Work verification experiment steps 1~4), bacterial infection is performed, the medium is removed, cells are taken, and lipid peroxidation detection is performed using the lipid peroxidation detection kit.
4. Measurement of Cell Viability treated with Fer-1:
According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8 kit is used to detect cell viability. The cells are treated with Fer-1 in this experiment. The absorbance of A450 nm is determined under a microplate reader by incubating with cells for 2 h under a co-incubation condition under a light shield.
shRNA Experiments: The Experiments on Engineered Bacteria Mediated Gene Silencing
Bacterial Infection:
1. Inoculate cancer cells in a culture dish and culture for a period of time to achieve appropriate state
and density.
2. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion (37°C, 220 rpm, 12 h).
3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5 (OD600=0.5 equals 1.6×108 bacterial cells/mL).
4. At least 30 minutes before co-incubation of bacteria, replace the original culture medium of cancer cells with DMEM basic culture medium with 10% FBS without antibiotics.
5. Take an appropriate amount of bacterial solution (the amount of bacterial solution depends on the MOI value), centrifuge and wash the bacteria twice with 1× PBS, and resuspend with DMEM without antibiotics or serum.
6. Add diluted cell culture medium containing corresponding engineered bacteria to cancer cells at the required MOI, and co-incubate the bacteria and cancer cells at 37°C for 2 hours.
7. After co-incubation for 2 hours, wash the cancer cells twice with 1× PBS, and then wash them once with DMEM culture medium added with antibiotics.
8. Continue to culture cancer cells in an atmosphere saturated with 37°C, 5% CO2, and water vapor for 24 hours.
9. The effect of bacterial treatment can be observed through fluorescence microscopy, measured by quantitative real-time RT-PCR for mRNA levels, and displayed by cell proliferation assay.
2. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for expansion (37°C, 220 rpm, 12 h).
3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5 (OD600=0.5 equals 1.6×108 bacterial cells/mL).
4. At least 30 minutes before co-incubation of bacteria, replace the original culture medium of cancer cells with DMEM basic culture medium with 10% FBS without antibiotics.
5. Take an appropriate amount of bacterial solution (the amount of bacterial solution depends on the MOI value), centrifuge and wash the bacteria twice with 1× PBS, and resuspend with DMEM without antibiotics or serum.
6. Add diluted cell culture medium containing corresponding engineered bacteria to cancer cells at the required MOI, and co-incubate the bacteria and cancer cells at 37°C for 2 hours.
7. After co-incubation for 2 hours, wash the cancer cells twice with 1× PBS, and then wash them once with DMEM culture medium added with antibiotics.
8. Continue to culture cancer cells in an atmosphere saturated with 37°C, 5% CO2, and water vapor for 24 hours.
9. The effect of bacterial treatment can be observed through fluorescence microscopy, measured by quantitative real-time RT-PCR for mRNA levels, and displayed by cell proliferation assay.
RNA Extraction from Adherent Cells:
1. Discard the culture medium and clean once with PBS.
2. Add 1 mL RNA isolator to every 10 cm2 of cultured cells to fully cover the cell surface, and blow the cells off using a pipette.
3. Transfer the lysate into a 1.5 mL centrifuge tube, blow repeatedly with a pipette until it is fully lysed, and let it stand on ice for 5 minutes.
4. Add 1/5 volume to the above cracking solution (200 μL) chloroform, shake vigorously to form an emulsion, and let stand at 4℃ for 5 minutes.
5. Centrifuged at 11200 rpm and 4 ℃ for 15 min.
6. Carefully remove the centrifuge tube, and the solution is divided into three layers: the upper aqueous phase, the middle white layer, and the lower red organic phase. Carefully suck the upper layer into a new centrifuge tube.
7. Add equal volume of pre-cooled isopropanol, invert and mix well. Allow to stand at 4 ℃ for 10 minutes.
8. 11200 rpm, centrifuged at 4 ℃ for 10 minutes, white precipitates appeared.
9. Carefully discard the supernatant and add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Gently flick the bottom of the tube to suspend the sediment, inverting it several times. Allow to stand at room temperature for 3-5 minutes.
10. Centrifuge at 11200 rpm and 4 ℃ for 5 minutes, discard the supernatant.
11.Dry and precipitate at room temperature for 2-5 minutes in a clean environment (be careful not to excessively dry, otherwise RNA will be difficult to dissolve).
12. Add an appropriate amount of RNase free ddH2O to dissolve the precipitate, gently blow with a pipette, take a small amount for testing after complete dissolution, and store the rest at -80℃ or use for the next reverse transcriptional reaction.
2. Add 1 mL RNA isolator to every 10 cm2 of cultured cells to fully cover the cell surface, and blow the cells off using a pipette.
3. Transfer the lysate into a 1.5 mL centrifuge tube, blow repeatedly with a pipette until it is fully lysed, and let it stand on ice for 5 minutes.
4. Add 1/5 volume to the above cracking solution (200 μL) chloroform, shake vigorously to form an emulsion, and let stand at 4℃ for 5 minutes.
5. Centrifuged at 11200 rpm and 4 ℃ for 15 min.
6. Carefully remove the centrifuge tube, and the solution is divided into three layers: the upper aqueous phase, the middle white layer, and the lower red organic phase. Carefully suck the upper layer into a new centrifuge tube.
7. Add equal volume of pre-cooled isopropanol, invert and mix well. Allow to stand at 4 ℃ for 10 minutes.
8. 11200 rpm, centrifuged at 4 ℃ for 10 minutes, white precipitates appeared.
9. Carefully discard the supernatant and add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Gently flick the bottom of the tube to suspend the sediment, inverting it several times. Allow to stand at room temperature for 3-5 minutes.
10. Centrifuge at 11200 rpm and 4 ℃ for 5 minutes, discard the supernatant.
11.Dry and precipitate at room temperature for 2-5 minutes in a clean environment (be careful not to excessively dry, otherwise RNA will be difficult to dissolve).
12. Add an appropriate amount of RNase free ddH2O to dissolve the precipitate, gently blow with a pipette, take a small amount for testing after complete dissolution, and store the rest at -80℃ or use for the next reverse transcriptional reaction.
Reverse Transcription Total RNA:
Quantitative Real-time PCR:
Anaerobism Experiments: The Experiments of Validation of Anaerobic Promoter pnirB
1. Add 5 mL LB liquid medium and 50 μL bacteria solution to 12 mL culture cube, culture 5 h at 37℃, 220
rpm.
2. Pretreatment to create low-oxygen conditions.
(i) Prepare 100 g/L Na2SO3 solution using sterilized water. Sterile filtration using 0.2 μm filter membranes.
(ii) Prepare 5 mL LB liquid medium with 1 g/L Na2SO3. In the meanwhile, the culture medium should be pre-reduced by placing it in an anaerobic tank containing anaerobic bags at room temperature over 10 hours in advance, which can absorb dissolved oxygen in the culture medium thoroughly.
3. Centrifuge the bacteria at 3000×g at room temperate for 5 min. Discard the supernatant.
4. Resuspend bacteria and transfer it to the medium treated with hypoxia as soon as possible.
5. Place the 12 mL culture cube in an anaerobic tank containing an anaerobic bag. Shake overnight at 37℃.
6. Take 100 μL bacterial solution for Western blotting.
2. Pretreatment to create low-oxygen conditions.
(i) Prepare 100 g/L Na2SO3 solution using sterilized water. Sterile filtration using 0.2 μm filter membranes.
(ii) Prepare 5 mL LB liquid medium with 1 g/L Na2SO3. In the meanwhile, the culture medium should be pre-reduced by placing it in an anaerobic tank containing anaerobic bags at room temperature over 10 hours in advance, which can absorb dissolved oxygen in the culture medium thoroughly.
3. Centrifuge the bacteria at 3000×g at room temperate for 5 min. Discard the supernatant.
4. Resuspend bacteria and transfer it to the medium treated with hypoxia as soon as possible.
5. Place the 12 mL culture cube in an anaerobic tank containing an anaerobic bag. Shake overnight at 37℃.
6. Take 100 μL bacterial solution for Western blotting.
TA System Experiments: The Experiments of Validation of Toxin-antitoxin System
Measurement of Plasmid Stability:
1. BL21 (DE3) transformed with pET-RFP-lacI and pGEN plasmids were grown on LB-agar plates supplemented with kanamycin and ampicillin.
2. Pick a single colony into 250 mL LB liquid medium without antibiotics, which was cultured at 37℃, 220 rpm.
3. Every 12 hours, 10 μL of bacteria solution was taken and transferred to another 20 mL of fresh LB liquid medium, which was still cultured at 37℃, 220 rpm for the next 12-hour growing cycle.
4. Repeat the above operations. When the accumulated time reached 48 h and 72 h, the appropriate amount of the culture was plated on the non-antibiotic LB-agar plates.
5. After single colonies grew on non-antibiotic LB-agar plates, count the white colonies and red colonies on each plate separately. The number of white colonies on each plate was divided by the total number of colonies on the plate to obtain the loss rate of plasmid containing pET-RFP-lacI in 48 hours and 72 hours.
6. A certain number of single colonies were randomly picked from non-antibiotic LB-agar plates to LB liquid medium containing ampicillin and grown in a 37℃ incubator. The loss rate of pGEN plasmid in the corresponding culture time was obtained by dividing the number of colonies ungrown by the total number of colonies which were picked from non-antibiotic LB-agar plates.
2. Pick a single colony into 250 mL LB liquid medium without antibiotics, which was cultured at 37℃, 220 rpm.
3. Every 12 hours, 10 μL of bacteria solution was taken and transferred to another 20 mL of fresh LB liquid medium, which was still cultured at 37℃, 220 rpm for the next 12-hour growing cycle.
4. Repeat the above operations. When the accumulated time reached 48 h and 72 h, the appropriate amount of the culture was plated on the non-antibiotic LB-agar plates.
5. After single colonies grew on non-antibiotic LB-agar plates, count the white colonies and red colonies on each plate separately. The number of white colonies on each plate was divided by the total number of colonies on the plate to obtain the loss rate of plasmid containing pET-RFP-lacI in 48 hours and 72 hours.
6. A certain number of single colonies were randomly picked from non-antibiotic LB-agar plates to LB liquid medium containing ampicillin and grown in a 37℃ incubator. The loss rate of pGEN plasmid in the corresponding culture time was obtained by dividing the number of colonies ungrown by the total number of colonies which were picked from non-antibiotic LB-agar plates.
Measurement of BL21(DE3) Specific Growth Rates:
1. Separately take 10 μL of BL21 (DE3) bacteria and 10 μL of engineered BL21 (DE3) bacteria containing pET-RFP-lacI and pGEN from the glycerol preservation, and add to 1 mL LB liquid medium for expansion (37°C, 220 rpm, 2.5 h).
2. Take out 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium and 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium with kanamycin, shaking at 37°C, 220 rpm.
3. Measure the OD600 of the samples at 15 minutes intervals after record the 0-time absorbance.
2. Take out 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium and 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium with kanamycin, shaking at 37°C, 220 rpm.
3. Measure the OD600 of the samples at 15 minutes intervals after record the 0-time absorbance.
Validation of T7-lac operator and pLtetO:
1. Separately take 10 μL of BL21 (DE3) bacteria containing the pET-GFP-lacI plasmid and 10 μL of original BL21 (DE3) bacteria from the glycerol preservation and add into 2 mL LB liquid medium for expansion (37°C, 200 rpm, 12 h).
2. Transfer 20 μL of BL21 (DE3) pET-GFP-lacI-containing bacteria solution into 2 mL of LB liquid medium containing kanamycin antibiotic (1:100) in six 12 mL bacteria-culturing tubes and 20 μL of BL21 (DE3) bacteria solution into 2 mL of LB liquid medium in three 12 mL bacteria-culturing tubes, culture at 37℃ until OD600=0.5 (OD600=0.5 equals to approximately 1× 108 CFU/mL).
3. Add 0.1 mM IPTG into three of the BL21 (DE3) pET-GFP-lacI-containing bacteria solution, culture for 6 h at 37℃, 220 rpm.
4. Measure the GFP fluorescence of nine bacteria solutions.
5. Validation of pLtetO with pJKR-L-tetR engineerd BL21 (DE3) bacteria in the same way with doxycycline.
2. Transfer 20 μL of BL21 (DE3) pET-GFP-lacI-containing bacteria solution into 2 mL of LB liquid medium containing kanamycin antibiotic (1:100) in six 12 mL bacteria-culturing tubes and 20 μL of BL21 (DE3) bacteria solution into 2 mL of LB liquid medium in three 12 mL bacteria-culturing tubes, culture at 37℃ until OD600=0.5 (OD600=0.5 equals to approximately 1× 108 CFU/mL).
3. Add 0.1 mM IPTG into three of the BL21 (DE3) pET-GFP-lacI-containing bacteria solution, culture for 6 h at 37℃, 220 rpm.
4. Measure the GFP fluorescence of nine bacteria solutions.
5. Validation of pLtetO with pJKR-L-tetR engineerd BL21 (DE3) bacteria in the same way with doxycycline.
References
1 Yu C, Zhai CK, Liao CS, et al. Construction and characterization of type III secretion system of
attenuated Salmonella typhimurium. Chin J Biotech, 2016, 32(12): 1664–1675.
2 Liu Z, Liu X, Cao W, Hua ZC. Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma. Am J Cancer Res. 2015 Jan 15;5(2):792-801. PMID: 25973316; PMCID: PMC4396039.
2 Liu Z, Liu X, Cao W, Hua ZC. Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma. Am J Cancer Res. 2015 Jan 15;5(2):792-801. PMID: 25973316; PMCID: PMC4396039.
